Mercapto-modified biocompatible macromolecule derivatives with low degree of mercapto-modification and the cross-linked materials and uses thereof

ABSTRACT

The present invention discloses a mercapto-modified biocompatible macromolecule derivative with a low degree of modification. The mercapto-modified biocompatible macromolecule derivative not only maintains the initial structure, physiological function and biocompatibility as much as possible, but also allows the preparation of the biocompatible macromolecule cross-linked material with a low degree of cross-linking through the effectively chemical cross-linking with the introduced mercapto group. The present invention further discloses a disulfide-bond cross-linked biocompatible macromolecule material with a very low degree of cross-linking. The disulfide-bond cross-linked biocompatible macromolecule material not only maintains the initial structure, physiological function and biocompatibility of the biocompatible macromolecule as much as possible, but also effectively prolongs turn over and reduces the solubility of the biocompatible macromolecule in vivo, better meeting the requirements of various clinical applications. The present invention further relates to the application of the disulfide-bond cross-linked biocompatible macromolecule material in the field of medicine and pharmacy.

TECHNICAL FIELD

The present invention relates to a biocompatible macromoleculederivative with a low degree of modification, and particularly to amercapto-modified biocompatible macromolecule derivative with a lowdegree of mercapto-modification; the present invention further relatesto a disulfide-bond cross-linked biocompatible macromolecule materialwith a low degree of cross-linking, and in addition further to the useof this cross-linked material in the field of medicine.

BACKGROUND ART

Biocompatible macromolecules have many important physiologicalfunctions, such as the significant effects of hyaluronic acid invisco-supplement treatment of osteoarthritis, wound healing promotionetc. However, the biocompatible macromolecules are usually turned oververy quickly in vivo or easily dissolved in the body fluid, whichlargely limits their uses in many medical applications. For example, thecourse of visco-supplement treatment of hyaluronic acid forosteoarthritis is a knee injection every week for five consecutiveweeks, which is inconvenient for patients and medical workers and alsoincreases the risk of infection. The chemical modification,cross-linking or crosslinking after modification is the effective methodfor biocompatible macromolecules to prolong their turn over and reducetheir solubility in vivo, which significantly expands their applicationsin clinical medicine. For example, as for the visco-supplement treatmentof osteoarthritis, the efficacy of one knee injection with thecross-linked sodium hyaluronate is equal to five knee injections withthe non-cross-linked sodium hyaluronate; besides, the cross-linkedhyaluronic acid has also been widely used for cosmetic purpose such asdermal fillers.

Although the application of the biocompatible macromolecules in theclinical medicine has greatly been expanded through their chemicalmodification and/or cross-linking, there are still conflicts betweentheory and practical processes. On one hand, to prolong their turn overand reduce their solubility in vivo the biocompatible macromoleculesshould be chemical modified/cross-linked to a certain degree. Thereforeall those chemically modified and/or cross-linked biocompatiblemacromolecule derivatives or cross-linked materials, which is widelyapplied in the clinical medicine currently, have a very high orrelatively high degree of modification or cross-linking, such as thehighly esterified derivative (up to 100% esterification) of sodiumhyaluronate (HYAFF, Fidia, Italy). On the other hand, the chemicalstructure of the biocompatible macromolecules is changed due to thechemical modification and/or cross-linking, which affects and reducestheir physiological function and biocompatible property and even causescertain side effects. For example, the study results reported by Jacobet al showed that MeroGel® (based on the highly modified HYAFF) causedinflammatory reaction and ossification reaction (Jacob et al.,Laryngoscope 112: 37-42, 2002).

However, most of the current research has focused on improving thedegree of modification and/or cross-linking to prolong turn over andreduce solubility of the biocompatible macromolecule in vivo. In ouropinion, the highly modified and/or cross-linked biocompatiblemacromolecule cannot better meet the requirements of the clinicalapplications in a considerable number of cases, and may even cause suchside effects as an inflammatory reaction etc. Therefore, the chemicalmodification and/or cross-linking of the biocompatible macromoleculemust be balanced between the following two factors: reducing the degreeof chemical modification and/or cross-linking as far as possible so asto maintain initial structure, physiological function andbiocompatibility, and meanwhile appropriately prolonging turn over andreducing solubility in vivo through chemical modification and/orcross-linking so as to meet the requirements of the clinicalapplications. However, it is a technical problem to balance the chemicalmodification and/or cross-linking of the biocompatible macromoleculebetween the above two factors.

The mercapto-modification and disulfide-bond cross-linking of thebiocompatible macromolecule is a new method of chemical modification andcross-linking, and has many advantages and thus many important potentialuses in the clinical medicine. For example, the mercapto-modifiedbiocompatible macromolecule derivatives have been used in chemicalactivity modification of various small molecular drugs and polypeptideprotein drugs, etc., and the cross-linked materials prepared based onthese mercapto-modified biocompatible macromolecule derivatives can beused as a cell growth matrix, a wound healing and regeneration matrix, adrug sustained-release carrier, a wound dressing, an in situ embeddingcell matrix, etc. (Bernkop-Schnurch, WO2000/025823; Shu et al.,Biomacromolecules, 3: 1304, 2002; Bulpitt et al., WO2002/068383;Prestwich et al., WO2004/037164; Prestwich et al., WO2005/056608;Prestwich et al., WO2008/008857; Song, WO2008/071058; Song,WO2008/083542; and Gonzalez et al., WO2009/132226). In general, it wasdeemed that a higher degree of mercapto-modification was needed for thepreparation of the subsequent cross-linked material of themercapto-modified biocompatible macromolecule derivative, and thereforein the above disclosed reference both the degree ofmercapto-modification and/or the degree of cross-linking of thebiocompatible macromolecule are very high, such as the Shu et al'sreport wherein 26.8%-66.8% of the groups were modified and cross-linked(Shu et al., Biomacromolecules, 3: 1304, 2002).

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram of the experimental results of Example 12of the present invention (i.e. a weight distribution diagram of a lefthindfoot).

SUMMARY

One aspect is related to a mercapto-modified biocompatible macromoleculederivative with a low degree of mercapto-modification, themercapto-modified biocompatible macromolecule derivative contains atleast three mercapto groups in its side chain, and have a degree ofmercapto-modification≦4.5%; the mercapto-modified biocompatiblemacromolecule derivative refers to a derivative obtained by chemicallyintroducing the mercapto group into the side-chain group of thebiocompatible macromolecule; the degree of mercapto-modification refersto a percentage of the amount of the introduced mercapto group in theamount of the available side-chain group of the biocompatiblemacromolecule for modification; and the biocompatible macromoleculerefers to a macromolecule having good biocompatibility, includingpolysaccharides, proteins, and synthetic macromolecules.

Another aspect is directed to a disulfide-bond cross-linkedbiocompatible macromolecule materials made from one or more of themercapto-modified biocompatible macromolecule derivatives with a lowdegree of mercapto-modification.

A further aspect is directed to use characterized in that the use in thefield of medicine includes a use in preparation of a postoperativeadhesion prevention formulation, a use in preparation of anosteoarthritis visco-supplement treatment formulation, and a use as asustained-release carrier of active therapeutic substances.

The aforementioned aspects and others are described in more detailbelow.

DETAILED DESCRIPTION

A technical problem to be solved by the present invention is to providea kind of mercapto-modified biocompatible macromolecule derivatives witha low degree of mercapto-modification. These mercapto-modifiedbiocompatible macromolecule derivatives maintains the initial structure,physiological function and biocompatibility of the originalbiocompatible macromolecule as much as possible, but also allows thepreparation of the biocompatible macromolecule cross-linked materialwith a low degree of cross-linking through the effectively chemicalcross-linking of the introduced mercapto group.

Another technical problem to be solved by the present invention is toprovide a disulfide-bond cross-linked biocompatible macromoleculematerial with a very low degree of disulfide-bond cross-linking. Thematerial of the invention not only have the initial structure,physiological function and biocompatibility of the originalbiocompatible macromolecule as much as possible, but also prolong theirturn over and reduce their solubility in vivo, better meeting therequirements of various medicine applications. Besides, thedisulfide-bond cross-linked biocompatible macromolecule material,allowing its cross-linking process to be completed in an injectablecontainer, is injectable, convenient to use, free of impurities,biocompatible, and free of toxic side effects, thus having very wideapplication prospects in the field of medicine.

Still another technical problem to be solved by the present invention isto provide a use of the above disulfide-bond cross-linked biocompatiblemacromolecule material in the field of medicine.

Some of the terms used in the present invention are defined as follows.

The biocompatible macromolecule refers to a macromolecule having goodbiocompatibility, including polysaccharides, proteins, syntheticmacromolecules, etc. Wherein the polysaccharides include chondroitinsulfate, dermatan, heparin, heparan, alginic acid, hyaluronic acid,dermatan sulfate, pectin, carboxymethyl cellulose, chitosan,carboxymethyl chitosan, etc., as well as the salts (e.g. sodium saltsand potassium salts) and derivatives thereof; the syntheticmacromolecules include polyacrylic acid, polyaspartic acid, polytartaricacid, polyglutamic acid, polyfumaric acid, etc., as well as the salts(e.g. sodium salts and potassium salts) and derivatives thereof; theproteins include collagen, alkaline gelatin, acidic gelatin, elastin,core protein, polysaccharide laminin, fibronectin, etc., as well as thesalts (e.g. sodium salts and potassium salts) and derivatives thereof.The biocompatible macromolecule is preferably chondroitin sulfate,heparin, heparan, alginic acid, hyaluronic acid, polyaspartic acid,polyglutamic acid, chitosan, carboxymethyl chitosan, alkaline gelatinand acidic gelatin, as well as the salts (e.g. sodium salts andpotassium salts) and derivatives thereof, and more preferablychondroitin sulfate and hyaluronic acid, as well as the salts (e.g.sodium salts and potassium salts) and derivatives thereof.

The mercapto-modified biocompatible macromolecule derivative refers to aderivative obtained by chemically introducing a mercapto group into theside-chain group of the biocompatible macromolecule; and the degree ofmercapto-modification refers to a percentage of the amount of theintroduced mercapto group in the amount of the available side-chaingroup for modification in the biocompatible macromolecule. For example,when the side-chain carboxyl group of the hyaluronic acid is subjectedto mercapto-modification, the degree of mercapto-modification refers toa percentage of the amount of the mercapto group in the total amount ofthe side-chain carboxyl group of the hyaluronic acid.

Disulfide-bond cross-linking refers to that the mercapto-modifiedbiocompatible macromolecule derivative forms a three-dimensionalreticular structure through the disulfide bond; and the degree ofdisulfide-bond cross-linking refers to a percentage of the amount of themercapto group of the mercapto-modified biocompatible macromoleculederivative forming the disulfide bond in the amount of the availableside-chain group for modification in the biocompatible macromolecule.

Hydrogel refers to a composite containing a great deal of water withthree-dimensional cross-linking network structure, which is betweenliquid and solid without fluidity. Gelation refers to a process throughwhich the liquid state with fluidity turns into the gel withoutfluidity.

Dynamic viscosity refer to the force for per unit area liquid requiredto move a unit distance at a unit velocity, which has a unit ofcentipoise (mPa·s) or poise (Pa·s). The dynamic viscosity is an indexfor assessing viscosity, the smaller the dynamic viscosity, the betterthe fluidity, and vice versa.

In one aspect, the present invention provides a mercapto-modifiedbiocompatible macromolecule derivative with a low degree ofmercapto-modification, which not only maintains the initial structure,physiological function and biocompatibility of the biocompatiblemacromolecule as much as possible, but also allows preparation of thebiocompatible macromolecule cross-linked material with a low degree ofcross-linking through the effectively chemical cross-linking with theintroduced mercapto group.

In the present invention, the mercapto-modified biocompatiblemacromolecule derivativewith a low degree of mercapto-modification canusually be prepared by the following methods, which have been describedin the patent document WO2009006780. A first method is the amino group(hydrazide)/carbodiimide coupling chemistry of the side-chain carboxylgroup. The usual way is as follows: The carboxyl group is activated bycarbodiimide to form an intermediate product that is followed bynucleophilic substitution with a disulfide-bond containing diamino ordihydrazide to produce another intermediate product, and finally thedisulfide-bond is reduced into a mercapto group to obtain themercapto-modified biocompatible macromolecule derivative (Shu et al.,Biomacromolecules, 3, 1304, 2002; Aeschlimann et al., U.S. Pat. No.7,196,180B1). A primary amine containing the free mercapto group (or amercapto-protected primary amine) can also be used instead of thedisulfide-bond containing diamino or dihydrazide to obtain themercapto-modified biocompatible macromolecule derivative or aintermediate product with mercapto protecting group that is deprotectedby removing the mercapto protecting group to obtain themercapto-modified biocompatible macromolecule derivative (Gianolio etal., Bioconjugate Chemistry, 16, 1512, 2005). The above carbodiimideusually refers to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride. A second method is to make the preparation through adirect reaction of the side-chain carboxyl group with the disulfide-bondcontaining carbodiimide (such as2,2′-dithiobis(N-ethyl-(N′-ethylcarbodiimide))), with the preparedmercapto-modified biocompatible macromolecule derivative having thestructure of the following formula (III) (Bulpitt et al., U.S. Pat. No.6,884,788?). A third method is to modify the side-chain amino group, andgenerally divided into two ways, i.e. direct and indirect modification.The direct modification method refers to introduce mercapto groupthrough the direct modification of the side-chain amino group, such asthe mercapto-modification of the collagen amino group by the activateddisuccinic bisacylcystamine dicarbonyl diimidazole ester (Yamauchi etal., Biomaterials, 22, 855, 2001; Nicolas et al., Biomaterials, 18, 807,1997). In the third method the indirect mercapto-modification of theamino group is generally divided into two steps. The first step iscarboxylation of the amino group, and the second step ismercapto-modification of the carboxyl group by the foregoing first orsecond methods. A fourth method is modification of the side-chainhydroxyl group. The usual way is that the hydroxyl group is carboxylatedin strong basic conditions, and then the carboxyl group ismercapto-modified in accordance with the foregoing first or secondmethods. For example, the side-chain hydroxyl group of suchmacromolecules as cellulose, hyaluronic acid, chitin and chitosan can becarboxymethylated, and is then mercapto-modified through the amino group(hydrazide)/carbodiimide chemical reaction.

For the biocompatible macromolecule with one or more kinds of functionalgroup (carboxyl group, amino group and hydroxyl group), the above one ormore methods can be adopted for the preparation of the mercapto-modifiedbiocompatible macromolecule derivative with a low degree ofmercapto-modification of the present invention.

In the present invention, the mercapto-modified biocompatiblemacromolecule derivative with a low degree of mercapto-modification isprepared by the foregoing preparation methods, and the present inventioncan then be carried out through adjustment of such parameters as thefeed ratio of the reaction materials, the reaction time and the reactiontemperature etc.

In the present invention, purification of the mercapto-modifiedbiocompatible macromolecule derivative with a low degree ofmercapto-modification is very important. Residual impurities may notonly produce toxic side effects such as inflammation in vivo, but alsointerfere with the subsequent disulfide-bond cross-linking. In thepresent invention, the residual impurities can be removed by dialysisand/or precipitation with organic solvent (e.g. ethanol) etc.

In the present invention, the adopted biocompatible macromolecule has amolecular weight in a range of 1,000-10,000,000 usually, preferably10,000-3,000,000, more preferably 20,000-1,500,000.

In the present invention, most of the initial structure of thebiocompatible macromolecule is retained, with a very low degree ofmercapto-modification. The mercapto-modified biocompatible macromoleculederivative with a low degree of mercapto-modification of the presentinvention contains at least three mercapto groups in its side chain,having a degree of mercapto-modification of ≦4.5%, preferably 0.5%-3.0%,more preferably 0.75%-2.5%.

Researchers generally have a technical prejudice to themercapto-modified biocompatible macromolecule derivative that a higherdegree of mercapto-modification is essential for the preparation of thesubsequent cross-linked material and meeting the requirements of theclinical applications. For example, Prestwich et al's researches showedthat only the biocompatible macromolecule derivative with a higherdegree of mercapto-modification could be cross-linked well (Prestwich etal., WO2008/008857). Therefore, researchers generally tend to improvethe degree of mercapto-modification of the biocompatible macromolecule.In 1983 Sparer et al. disclosed the derivatives of glycosaminoglycan(hyaluronic acid and chondroitin sulfate)-cysteine methyl ester, whereinthe cysteine methyl ester was coupled with the glycosaminoglycan via anamide bond, and 5%-87% of the side-chain carboxyl group of theglycosaminoglycan was modified into a mercapto group (Sparer et al.,Chapter 6, Pages 107-119, Controlled Release Delivery System, Edited byTheodore J. Roseman and S. Z. Mansdorf, Marcel Dekker Inc.). In 2005Gianolio et al. disclosed the hyaluronic acid-cysteamine derivative,wherein the cysteamine was coupled with the side-chain carboxyl group ofthe hyaluronic acid via an amide bond, and 22% of the side-chaincarboxyl group of the hyaluronic acid was modified into the mercaptogroup (Gianolio et al., Bioconjugate Chemistry, 16: 1512-1518, 2005). In2008 Yin et al. disclosed the hyaluronic acid-cysteamine derivative,wherein the cysteamine was coupled with the side-chain carboxyl group ofthe hyaluronic acid via an amide bond, the derivative contained both10-200 μmol/g mercapto group and 120-500 μmol/g disulfide bond, and thedegree of mercapto-modification was 10%-48% calculated based on that thedisaccharide repeating unit of the hyaluronic acid had a molecularweight of 400 (i.e. 10%-48% of the side-chain carboxyl group of thehyaluronic acid was mercapto-modified) (Yin et al., CN 101367884). Thehyaluronic acid mercapto-modified derivative coupled via the hydrazidebond disclosed by Shu et al. had a degree of mercapto-modification of26.8%-66.8% (Shu et al., Biomacromolecules, 3: 1304, 2002).

However, when the degree of mercapto-modification is high, the initialstructure of the biocompatible macromolecule is modified significantly,which may compromise its physiological function and biocompatibility.For example, Wang et al's research results showed that the chitosanmercapto-modified derivative produced significant cell toxicity at ahigh degree of mercapto-modification (Wang et al., Chemical Journal ofChinese universities, 29: 206-211, 2008). Our researches also showedthat the high degree of mercapto-modification changed the structure ofhyaluronic acid, and interfered in the binding with its receptor (e.g.CD44).

In the present invention, the prepared mercapto-modified biocompatiblemacromolecule derivative having a low degree of mercapto-modificationwas purified by the above one or more methods, with the residualimpurities usually less than 1/1,000 and even 1/10,000 (weight content).

The present invention has the following advantageous effects: Themercapto-modified biocompatible macromolecule derivative with a lowdegree of mercapto-modification of the present invention has a very lowdegree of mercapto-modification, not only maintaining the initialstructure, physiological function and biocompatibility of thebiocompatible macromolecule as much as possible, but also having suchfeatures as consuming little raw materials and costing a short reactiontime. Furthermore, the mercapto-modified biocompatible macromoleculederivative with a low degree of mercapto-modification of the presentinvention can be used conveniently in the preparation of thecross-linked materials and meeting the requirements of various clinicalapplications. Moreover, the present invention also overcomes theforegoing technical prejudice that a high degree ofmercapto-modification is essential for preparation of the subsequentcross-linked material of the mercapto-modified biocompatiblemacromolecule derivative and meeting the requirements of the clinicalapplications.

In another aspect, the present invention provides a disulfide-bondcross-linked biocompatible macromolecule material with a very low degreeof disulfide-bond cross-linking, which not only maintains the initialstructure, physiological function and biocompatibility of thebiocompatible macromolecule as much as possible, but also prolongs itsturnover and reduces solubility in vivo, better meeting the requirementsof various clinical applications. The disulfide-bond cross-linkedbiocompatible macromolecule material of the present invention is usuallypresent in a form of hydrogel, which has water content preferably ofmore than 95% (w/v, g/ml), and more preferably of more than 98% (w/v,g/ml). The disulfide-bond cross-linked biocompatible macromoleculehydrogel of the present invention can be made into various solid formssuch as film and sponge after being dried or freeze-dried.

The disulfide-bond cross-linked biocompatible macromolecule hydrogel ofthe present invention was made from the mercapto-modified biocompatiblemacromolecule derivative with a low degree of mercapto-modification ofthe present invention. A first method of preparation is as follows: Themercapto-modified biocompatible macromolecule derivative with a lowdegree of mercapto-modification of the present invention is dissolved inwater to obtain a solution of a suitable concentration (usually0.2%-5.0%), which is adjusted to a specific pH value (usually neutral,i.e. a pH value of about 7), and then the mercapto group is oxidizedunder the action of the oxygen in the air and the dissolved oxygen inthe solution to form the disulfide bond gradually, making the solutiongradually gelatinated and the dynamic viscosity of the solutiongradually increased, finally making the solution lose fluidity to form athree-dimensional cross-linked network structure. An oxidant (e.g.hydrogen peroxide) can further be added into the above solution toaccelerate the cross-linking process.

A second method of preparation of the disulfide-bond cross-linkedbiocompatible macromolecule hydrogel of the present invention is to usethe method disclosed by Shu et al. (WO2010043106). In this method, thegelation process can be completed in an injectable container. And thegel has the advantage of allowing injection, convenient use, noimpurities, good biocompatibility, no toxic side effects, etc. Thismethod is specifically as follows: The mercapto-modified biocompatiblemacromolecule derivative with a low degree of mercapto-modification ofthe present invention is dissolved in water to obtain a solution of asuitable concentration (usually 0.2%-5.0%), which is adjusted to aspecific pH value (usually neutral), and then the solution is filledinto the injectable container and sealed, with the mercapto groupgradually forming the disulfide-bond mainly under the action ofoxidation of the dissolved oxygen in the solution, making the solutiongradually gelatinated and the dynamic viscosity of the solutiongradually increased, finally making the solution lose fluidity to form athree-dimensional network cross-linked structure. An oxidant (e.g.hydrogen peroxide) can further be added into the above solution toaccelerate the cross-linking process.

An aseptic process or a terminal sterilization process (e.g. the moistheat sterilization process commonly used in the pharmaceutical industry)can be adopted in the production when the second method of preparationof the disulfide-bond cross-linked biocompatible macromolecule hydrogelof the present invention is adopted, so as to meet differentrequirements of clinical medicine. The filling production line commonlyused in the pharmaceutical industry can be used to realize thelarge-scale industrialized production, with the hourly output easilyamounting to more than 3000 pieces. The filling production line can beselected from a straight line full-automatic syringe prefillingproduction line or a beehive syringe full-automaticprefilling-and-plugging machine manufactured by the Groninger company,and a presterilized syringe liquid filling machine manufactured by theBosch company of Germany, etc. The injectable container can be a syringemade of glass or plastics, such as the Hypak SCF presterilizationsyringe manufactured by BD company, and the syringe can also be replacedby such extrusible containers as a soft plastic bag.

In the disulfide-bond cross-linked biocompatible macromolecule hydrogelof the present invention, the mercapto-modified biocompatiblemacromolecule derivative having a low degree of mercapto-modification ofthe present invention is used as the raw material, and the degree ofdisulfide-bond cross-linking is dependent on the degree ofmercapto-modification (≦4.5%), therefore the disulfide-bond cross-linkedbiocompatible macromolecule hydrogel of the present invention also has avery low degree of disulfide-bond cross-linking (≦4.5%). Usually morethan half of the mercapto groups in the disulfide-bond cross-linkedbiocompatible macromolecule hydrogel of the present invention areoxidized into the disulfide bond, which results in the formation of thethree-dimensional cross-linked network structure, loss of fluidity ofthe liquid solution, and the very high dynamic viscosity. Compared withthe non-crosslinked solution, the dynamic viscosity of thedisulfide-bond cross-linked biocompatible macromolecule hydrogel of thepresent invention is usually increased by more than 50 times, and caneven be increased by more than 500 times under optimal conditions. Thedisulfide-bond cross-linked biocompatible macromolecule hydrogel of thepresent invention has such characteristics that it has a uniqueadvantage in such important clinical applications as the prevention andcontrol of postoperative adhesion, and the osteoarthritisvisco-supplement treatment.

The dynamic viscosity of the disulfide-bond cross-linked biocompatiblemacromolecule hydrogel of the present invention was measured with arotation viscometer at a shear rate of not less than 0.25 Hz and atemperature of 25±0.1 according to the second method in Appendix VI G ofthe Pharmacopoeia of the People's Republic of China (second part, 2005Edition), and is typically more than 10,000 centipoise (mPa·s),preferably greater than 25,000 centipoise (mPa·s), and more preferablygreater than 40,000 centipoise (mPa·s).

The disulfide-bond cross-linked biocompatible macromolecule material ofthe present invention may contain one or more mercapto-modifiedbiocompatible macromolecule derivatives with a low degree ofmercapto-modification of the present invention, as well as one or moreother substances. These substances can be polysaccharides, proteins orsynthetic macromolecules, such as chondroitin sulfate, heparin, heparan,alginic acid, hyaluronic acid, polyaspartic acid, polyglutamic acid,chitosan, carboxymethyl chitosan, collagen, alkaline glutin and acidicglutin, as well as the salts (e.g. sodium salts and potassium salts) andderivatives thereof, preferably sodium hyaluronate, chondroitin sulfate,heparin sodium, alkaline glutin and acidic glutin, etc., and morepreferably sodium hyaluronate, chondroitin sulfate and heparin sodium;these substances can also be active ingredients, including steroids,antibiotics, drugs for the treatment of tumors, and various polypeptideprotein drugs such as cortical hormones (of steroids), e.g.beclomethasone, beclomethasone dipropionate, budesonide, dexamethasone,prednisolone, and prednisone; again such as various polypeptide proteindrugs, e.g. various growth factors (an alkaline growth factor, an acidicgrowth factor, a blood vessel growth factor, an ossification growthfactor, etc.), and nucleic acids (e.g. RNA). These active ingredientscan be dispersed and/or dissolved in a form of solid particles in thedisulfide-bond cross-linked biocompatible macromolecule material of thepresent invention.

In the actual application in the field of medicine, it is required thatthe disulfide-bond cross-linked biocompatible macromolecule hydrogelshould have an appropriate shelf-life, and its properties should bestable. However, the disulfide-bond cross-linked biocompatiblemacromolecule hydrogel having a high degree of modification is notstable, the hydrogel gradually contracts such that a large amount ofwater is extruded from the hydrogel with the increase of the storagetime, which makes the dynamic viscosity greatly reduced and seriouslyaffects the gel properties, not meeting the needs of practical clinicalapplications and seriously restricting application of the disulfide-bondcross-linked biocompatible macromolecule hydrogel in the field ofmedicine. For example, the volume of hydrogel contracts about 30% afterthe disulfide-bond cross-linked hyaluronic acid hydrogel (having adegree of mercapto-modification of 13.5%) has been stored at roomtemperature for six months.

The disulfide-bond cross-linked biocompatible macromolecule hydrogel ofthe present invention was made from the mercapto-modified biocompatiblemacromolecule derivative with a low degree of mercapto-modification ofthe present invention, the unexpected technical effects was achieved,and the above problem of instability of the disulfide-bond cross-linkedbiocompatible macromolecule hydrogel was solved. The six-monthaccelerated stability tests showed that the disulfide-bond cross-linkedbiocompatible macromolecule hydrogel of the present invention has goodstability, which will further be described with reference to examples.

The present invention has the following advantageous effects: Thedisulfide-bond cross-linked biocompatible macromolecule material of thepresent invention, having a very low degree of cross-linking, not onlymaintains the initial structure, physiological function andbiocompatibility of the biocompatible macromolecule as much as possible,but also has the very high dynamic viscosity, effectively prolongs theturn over and reduces the solubility of the biocompatible macromoleculein vivo, better meets the requirements of various clinical applications.The present invention further has the following advantageous technicaleffect that the disulfide-bond cross-linked biocompatible macromoleculehydrogel of the present invention has good stability.

In other aspect, the present invention further provides the applicationof the above disulfide-bond cross-linked biocompatible macromoleculematerial in the field of medicine.

The applications of the disulfide-bond cross-linked biocompatiblemacromolecule material of the present invention in medicine include thefollowing aspects: it can be used as wound dressing for skin or otherwounds to promote wound healing; it can also be used for preventingadhesion, including the fibrous adhesion between tissues or organs afterthe surgery (e.g. sinusitis surgery); it can also be used in theosteoarthritis visco-supplement treatment as a knee lubricant.

The applications of the disulfide-bond cross-linked biocompatiblemacromolecule material prepared by the present invention in pharmacyinclude that it can be used as a sustained-release carrier for variousactive therapeutic substances to realize sustained release. The activetherapeutic substances may be a chemical drug or a biologically activefactor, including antiphlogistics, antibiotics, analgesics, anesthetics,wound healing promoters, cell growth promoters or inhibitors, immunestimulants, antiviral drugs, etc.

The present invention has at least the following advantageous effects:The mercapto-modified biocompatible macromolecule derivative with a lowdegree of mercapto-modification of the present invention has a very lowdegree of mercapto-modification, not only maintaining the initialstructure, physiological function and biocompatibility of thebiocompatible macromolecule as much as possible, but also having suchfeatures as consuming little raw material and costing a short reactiontime. Furthermore, the mercapto-modified biocompatible macromoleculederivative with a low degree of mercapto-modification of the presentinvention can be used conveniently in preparation of the cross-linkedmaterials and meeting the requirements of various clinical applications.Moreover, the present invention also overcomes the foregoing technicalprejudice that a high degree of mercapto-modification is essential forpreparation of the subsequent cross-linked material of themercapto-modified biocompatible macromolecule derivative and to meet therequirements of the clinical applications.

The present invention has at least the following advantageous effects:The disulfide-bond cross-linked biocompatible macromolecule material ofthe present invention, having a very low degree of cross-linking, notonly maintains the initial structure, physiological function andbiocompatibility of the biocompatible macromolecule as much as possible,but also has the very high dynamic viscosity, effectively prolongs theturn over and reduces the solubility of the biocompatible macromoleculein vivo, better meets the requirements of various clinical applications.The present invention further also has the following advantageoustechnical effect that the disulfide-bond cross-linked biocompatiblemacromolecule hydrogel of the present invention has good stability.

EXAMPLES

The following examples can make those skilled in the art understand thepresent invention more completely, rather than limit the presentinvention in any way.

Example 1 Preparation and Characterization of the Mercapto-ModifiedHyaluronic Acid Derivative

The preparation was made according to the method disclosed by Shu et al.in Biomacromolecules, 3, 1304, 2002. Dithiodipropionic dihydrazide wasadded to a solution of hyaluronic acid (11.9 g) in distilled water (2L). The mixture was stirred until dissolved. Then after pH value of thesolution was adjusted to 4.75 with 0.1 mol/L hydrochloric acid, acertain amount of 1-ethyl-3-(3-dimethylaminepropyl) carbodiimidehydrochloride (EDCI) (Aldrich, the United States) was added according toTable 1 under electromagnetic stirring. An amount of 0.1 mol/Lhydrochloric acid was added continuously into the above solution to keepthe solution at pH 4.75. The reaction was terminated by adding 1.0 mol/Lsodium hydroxide to adjust the pH value to 7.0. Then 100 gdithiothreitol (Diagnostic Chemical Limited, the United States) and anamount of 1.0 mol/L sodium hydroxide were added with stirring. pH valueof the solution was adjusted to 8.5. The reaction was electromagneticstirred at room temperature for 24 hours. Then 1 mol/L hydrochloric acidwas added into the above solution until pH 3.0. The above solution wasloaded into a dialysis tube (the molecular-weight cutoff (MWCO) of3,500, Sigma, the United States), and was dialyzed for 5 days against agreat deal of 0.001 mol/L hydrochloric acid and 0.2 mol/L sodiumchloride, with the dialys ate changed every 8 hours; then the solutionwas dialyzed again for 3 days against a great deal of 0.001 mol/Lhydrochloric acid, with the dialysate changed every 8 hours. Finally thesolution in dialysis tube was collected for direct application orfreeze-dried to givewhite flocculent solid.

The content of the mercapto group was detected by the modified Ellmanmethod reported by Shu et al. in Biomacromolecules, 3, 1304, 2002 andthe degree of mercapto-modification was calculated, or the degree ofmercapto-modification was measured by using the hydrogen spectrumnuclear magnetic resonance (¹H-NMR) (with D₂O as the solvent) (takingthe characteristic methyl group absorption peak of the acetyl group ofhyaluronic acid as the internal standard). The degree ofmercapto-modification refers to a percentage of the amount of themercapto group in the total amount of the side-chain carboxyl group ofthe hyaluronic acid, with the measurement results as follows:

TABLE 1 Degree of mercapto-modification Serial number 1 2 3 4 5 6 7 8 9EDCI 0.2  0.3  0.4  0.6  0.8  1.0  1.2   2.4 9.6 feeding amount (g)Degree of 0.48 1.04 1.46 2.33 3.24 4.18 4.61 10.6 37 mercapto- modif -ication (%)

Example 2 Preparation and Characterization of the Mercapto-ModifiedChondroitin Sulfate Derivative

1 g chondroitin sulfate (Type c, from the shark cartilage, Sigma, theUnited States) was dissolved in 100 ml distilled water to give a clearand transparent solution. To the solution 0.6 g dithiodipropionicdihydrazide was added. The mixture was stirred until dissolved. Then pHvalue of the solution was adjusted to 4.75 with 0.1 mol/L hydrochloricacid, and a certain amount of 2-ethyl-3-(3-dimethylaminepropyl)carbodiimide hydrochloride (EDCI) (Aldrich, the United States) was addedaccording to Table 2 under electromagnetic stirring. An amount of 0.1mol/L hydrochloric acid continuously was added into the above solutionto keep the solution at pH 4.75. The solution was stirredelectromagnetically for 2 hours at room temperature. Then 10 gdithiothreitol (Diagnostic Chemical Limited, the United States) and alittle of 0.1 mol/L sodium hydroxide was added with stirring. Meanwhile,0.1 mol/L sodium hydroxide was added continuously to keep the solutionat pH 8.5, and the solution was stirring electromagnetically for 4 hoursat room temperature. Then 6 mol/L hydrochloric acid was into the abovesolution until pH 3.0. The above solution was loaded into a dialysistube (of the MWCO of 2,000, Sigma, the United States), and was dialyzedfor 5 days against 2 L solution of hydrochloric acid (0.001 mol/L) andsodium chloride (0.3 mol/L), with the dialysate changed every 8 hours;then the solution was dialyzed again for 3 days against 2 L hydrochloricacid (0.001 mol/L), with the dialysate changed every 8 hours. Finallythe solution in dialysis tube was collected for direct application orfreeze-dried to give white flocculent solid.

The content of the mercapto group was detected by the modified Ellmanmethod reported by Shu et al. in Biomacromolecules, 3, 1304, 2002 andthe degree of mercapto-modification was calculated, or the degree ofmercapto-modification was measured by using the hydrogen spectrumnuclear magnetic resonance (¹H-NMR) (with D₂O as the solvent) (takingthe characteristic methyl group absorption peak of the acetyl group ofchondroitin sulfate as the internal standard). The degree ofmercapto-modification refers to a percentage of the amount of themercapto group in the total amount of the side-chain carboxyl group ofthe chondroitin sulfate, with the measurement results as follows:

TABLE 2 Degree of mercapto-modification Serial number 1 2 3 4 5 6 7 8 9EDCI 0.01 0.015 0.02 0.03 0.04 0.05 0.06  0.12  0.48 feeding amount (g)Degree of 0.88 1.54  1.96 3.33 4.50 5.18 6.81 15.6  42.1  mercapto-modi- fication (%)

Example 3 Preparation and Characterization of the Mercapto-ModifiedHyaluronic Acid Derivative

Sodium salt of Sulfo-N-hydroxy succinimide (Sulfo-NHS), cystaminedihydrochloride (CYS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDCI) was added to a solution of hyaluronic acid (10 g)in distilled water (1 L) respectively according to the amounts in Table3. The mixture was stirred until dissolved. Then the pH value of thesolution was adjusted to 4.5-6.5 with 0.1 mol/L hydrochloric acid underelectromagnetic stirring to react for a period of time. An amount of 0.1mol/L hydrochloric acid continuously was added into the above solutionto keep the solution at pH 4.5-6.5. The reaction was terminated byadding 1.0 mol/L sodium hydroxide to adjust the pH value to 8.5. Then 50g dithiothreitol (Diagnostic Chemical Limited, the United States) and anamount of 1.0 mol/L sodium hydroxide were added with stirring. The pHvalue of the solution was adjusted to 8.5. The solution was stirredelectromagnetically for 24 hours at room temperature. Then 1 mol/Lhydrochloric acid was added into the above solution until pH 3.0. Theabove solution was loaded into a dialysis tube (the MWCO of 3,500,Sigma, the United States), and was dialyzed for 5 days against a greatdeal of 0.001 mol/L hydrochloric acid and 0.5 mol/L sodium chloride,with the dialysate changed every 8 hours; then the solution was dialyzedagain for 3 days against a great deal of 0.001 mol/L hydrochloric acid,with the dialysate changed every 8 hours. Finally the solution indialysis tube was collected for direct application or freeze-dried togive white flocculent solid.

The content of the mercapto group was detected by the modified Ellmanmethod reported by Shu et al. in Biomacromolecules, 3, 1304, 2002 andthe degree of mercapto-modification was calculated, or the degree ofmercapto-modification was measured by using the hydrogen spectrumnuclear magnetic resonance (¹H-NMR) (with D₂O as the solvent) (takingthe characteristic methyl group absorption peak of the acetyl group ofhyaluronic acid as the internal standard). The degree ofmercapto-modification refers to a percentage of the amount of themercapto group in the total amount of the side-chain carboxyl group ofthe hyaluronic acid, with the measurement results as follows:

TABLE 3 Degree of mercapto-modification Serial number 1 2 3 4 5 6 7 8 9Sulfo-NHS (g) 5.43 21.62 5.43 11.62 2.72 10.86 21.62 5.43 5.43 CYS (g)22.6 11.3 33.9 17 5.625 22.6 11.3 22.6 11.3 EDCI (g) 4.8 9.6 4.8 2.4 2.41.2 1.2 2.4 0.72 Reaction time (Hour) 12 3 1 1 1 1 8 8 8 Degree ofmercapto- 13.5 4.83 1.54 0.84 0.73 0.51 2.14 3.97 1.28 modification (%)

Example 4 Preparation of the Disulfide-Bond Cross-Linked Hyaluronic AcidHydrogel

The two kinds of mercapto-modified hyaluronic acid derivative preparedin Example 1 (having a degree of mercapto-modification of 2.33% and4.18%, and indicated as Nos. 4 and 6 in Table 1, respectively) weredissolved to give a 10 mg/ml solution, a 15 mg/ml solution and a 20mg/ml solution, respectively, with the pH values adjusted to 7.4. Theabove solutions (2 ml) were transferred into 10 ml glass bottles andsealed respectively, and stand at room temperature for one week. Thusthe solutions lose their fluidity and form the cross-linked hydrogels,with the water content of the hydrogels (g/ml) respectively being 99%,98.5% and 98%.

Example 5 Preparation of the Disulfide-Bond Cross-Linked ChondroitinSulfate Hydrogel

The mercapto-modified chondroitin sulfate derivative prepared in Example2 (having a degree of mercapto-modification of 4.50%, and indicated asNo. 5 in Table 2) was dissolved to give a 50 mg/ml solution and an 80mg/ml solution, respectively, with the pH values adjusted to 7.4. Theabove solutions (2 ml) were transferred into 10 ml glass bottles andsealed respectively, and stand at room temperature for one week. Thusthe solutions lose their fluidity and form the cross-linked hydrogels,with the water content of the hydrogels (g/ml) respectively being 95%and 92%.

Example 6 Preparation of the Disulfide-Bond Cross-Linked Hyaluronic AcidHydrogel

The four kinds of mercapto-modified hyaluronic acid derivative preparedin Example 3 (having a degree of mercapto-modification of 1.28%, 1.54%,2.14% and 3.97%, and indicated as Nos. 3, 7, 8 and 9 in Table 3,respectively) were dissolved to give a 5 mg/ml solution, a 7.5 mg/mlsolution and a 10 mg/ml solution, respectively, with the pH valuesadjusted to 7.4. The above solutions (2 ml) were transferred into 10 mlglass bottles and sealed respectively, and stand at room temperature for10 days. Thus the solutions lose their fluidity and form thecross-linked hydrogels, with the water content of the hydrogels (g/ml)respectively being 99.5%, 99.25% and 99%.

Example 7 Preparation of the Disulfide-Bond Cross-Linked Hyaluronic AcidHydrogel

The mercapto-modified hyaluronic acid derivative prepared in Example 3(having a degree of mercapto-modification of 2.14%, and indicated as No.7 in Table 3) was dissolved to give a 10 mg/ml solution, and then thehyaluronic acid solution (5 mg/ml) and the chondroitin sulfate solution(10 mg/ml) were added according to a volume ratio of 2:1, respectively,with the pH value adjusted to 7.4. 2 ml of the above solution wastransferred into a 10 ml glass bottle and sealed, and stand at roomtemperature for 10 days. Thus the solution loses its fluidity and formsthe cross-linked hydrogel.

Example 8 Measurement of Dynamic Viscosity of the Disulfide-BondCross-Linked Hyaluronic Acid Hydrogel

The dynamic viscosity of the disulfide-bond cross-linked hyaluronic acidhydrogel prepared in Example 6 was measured with a rotation viscometerat a shear rate of not less than 0.25 Hz and a temperature of 25±0.1according to the second method in Appendix VI G of volume II,Pharmacopoeia of the People's Republic of China (2005 Edition), with theresults as shown in Table 4. The dynamic viscosity of the cross-linkedhydrogel was increased by 408-547 times compared to the correspondingunmodified hyaluronic acid solution.

TABLE 4 Dynamic viscosity (mPa · s) Concentration Degree of Degree ofDegree of Degree of of Degree of mercapto- mercapto- mercapto- mercapto-hyaluronic mercapto- modification modification modification modificationacid modification (%) (%) (%) (%) (mg/ml) (%) 0 1.28 1.54 2.14 3.97 5137 75000 78000 81000 88000 7.5 198 >100000 >100000 >100000 >100000 10245 >100000 >100000 >100000 >100000

Example 9 Preparation and Stability Test of the Disulfide-BondCross-Linked Hyaluronic Acid Hydrogel

Hydrogel 1: The mercapto-modified hyaluronic acid derivative prepared inExample 3 (having a degree of mercapto-modification of 13.5%, andindicated as No. 1 in Table 3) was dissolved to give a 10 mg/mlsolution, with the pH value adjusted to 7.4. 2 ml of the above solutionwas transferred into a 10 ml glass bottle and sealed, and stand at roomtemperature for 10 days. Thus the solution loses its fluidity and formsthe cross-linked hydrogel.

Hydrogel 2: The hydrogel prepared in Example 6 (the hyaluronic acidmercapto-modified derivative prepared in Example 3, having aconcentration of hyaluronic acid of 10 mg/ml and a degree ofmercapto-modification of 1.54%, and indicated as No. 3 in Table 3).

It is thus clear that the degree of mercapto-modification of the rawmaterial of Hydrogel 2 (i.e. 1.54%) is obviously lower than that of theraw material of Hydrogel 1 (i.e. 13.5%), that is, the degree ofcross-linking of Hydrogel 2 is obviously lower than that of Hydrogel 1.

Stability test: An accelerated stability test was performed on thehydrogels according to the Guiding Principle of Stability Test of DrugSubstances and Drug Product as provided in XIX C of volume II, ChinesePharmacopoeia 2010 edition, with the temperature kept at 40±2 for 6months; sampling and measuring the dynamic viscosity and contractionpercentage (%) of the hydrogel at the end of 0, 1, 2, 3 and 6 monthsduring the test, with the results as shown in Table 5. For Hydrogel 1having a high degree of cross-linking, with the accelerated stabilitytests conducted, the dynamic viscosity declined sharply and the volumeof the gel decreased consecutively, the volume of the gel having adecreasing percentage of 10.2%, 35.1%, 39.2% and 41.4% respectivelyafter 1, 2 and 3 and 6 months, with a great deal of water extruded fromthe gel. While Hydrogel 2 having a low degree of cross-linking of thepresent invention kept a good stability.

TABLE 5 Stability test results Time Time Time Time Time (month) (month)(month) (month) (month) 0 1 2 3 6 Hydro- Dynamic >100000 8750 <5000<5000 <5000 gel 1 viscosity Hydro- Contraction 0 10.2 35.1 39.2 41.4 gel1 percentage (%) Hydro- Dynamic >100000 >100000 >100000 >100000 >100000gel 2 viscosity Hydro- Contraction 0 0 0 0 0 gel 2 percentage (%)

Example 10 The Disulfide-Bond Cross-Linked Hyaluronic Acid HydrogelPreventing the Sinus Ostium Stenosis after the Sinusitis Surgery

10 male pasteurized New Zealand white rabbits with a weight of 3.5-4.0kg were anesthetized by intramuscular injection of ketamine (35 mg/kg)and toluolzosin (5 mg/kg). After peeling off external backside of theirnoses, the rabbits were disinfected with iodine, and then anesthetizedwith a mixed liquid of 3 ml of 1% lidocaine and 1:100,000 adrenaline.Under aseptic conditions, a 2.5 mm perpendicular incision was made alongthe midline, and the soft tissues and the periosteum covered on thegenyantrum were lifted and separated. The anterior wall of thegenyantrum was opened with an electric surgical drill, and brokenthrough between middle wall of the genyantrum and the nasal cavity witha 4 mm spherical cutting drill, thus forming a cylindrical ostium of 4mm in diameter without mucosa on the edge. 5 rabbits at their both sidesof the ostium were filled with the hydrogel prepared in Example 6(having a concentration of hyaluronic acid of 10 mg/ml, and a degree ofmercapto-modification of 1.54%) (the treated group), and the other 5rabbits at their both sides of the ostium was filled nothing (thecontrol group). Then the periosteum was sutured interruptedly with anabsorbable suture, and the skin was sutured with an absorbable suture toseal the genyantrum. No other dressing was used. The animals were fedwith normal diet and drinking water after the operation.

The rabbits were killed after two weeks. The healed wound was incisedafter the killing to expose the sinus cavity. The residue in the sinuscavity was flushed with water and sucked gently with an extractor. Themedial wall of the sinus was inspected with a 30-degree nasal endoscopeand recorded. Each of the ostium was measured with a ruler of millimeterscale. The ostium was observed and measured by the double-blind method.The ostium in the treated group had a diameter of 2.78±1.17 mm, whilethe ostium of the control group had a diameter of 0.7±0.52 mm.

The stenosis of the ostium, as an important problem with the sinusitisclinical surgery, will affect the surgical effect, and even cause thesinusitis relapse. The above results indicate that the disulfide-bondcross-linked hyaluronic acid hydrogel having a low degree ofcross-linking of the present invention can significantly prevent theostium from stenosis, and is thus expected to have wide applications inclinics.

Example 11 Application of the Disulfide-Bond Cross-Linked HyaluronicAcid Hydrogel in the Postoperative Adhesion Prevention

The rat cecum model reported by Hemadeh et al. (Surgery 114: 907-10,1993) and Yetkin et al. (Int J Surg 7: 561-65, 2009) was used. Theprocess is summarized as follows: 32 rats were divided into 3 groups,with the serosa luster of their cecum serosa scraped off using sterilegauze until the surface bleeding; then a drop of anhydrous ethanol wasdropped to the bleeding surface to induce further adhesion; Group 1 wasa control group without any treatment, Group 2 was treated with 1 mlcommercially available hyaluronic acid solution (10 mg/ml), and Group 3was treated with the hydrogel prepared in Example 6 (having aconcentration of hyaluronic acid of 10 mg/ml and a degree ofmercapto-modification of 1.54%); finally the surface wound of the ratswas sutured. after two weeks the rats were killed and dissected toobserve the adhesion status.

The adhesion was evaluated according to the Yetkin et al's adhesionevaluation system (Int J Surg 2009; 7: 561-65), with the results asshown in Table 6. The blank control group (Group 1) had severe adhesion,the commercially available hyaluronic acid therapeutic group (Group 2)had a certain degree of adhesion, and the disulfide-bond cross-linkedhyaluronic acid hydrogel of the present invention (Group 3) had the besteffects in adhesion prevention.

TABLE 6 Adhesion score Group 1 Group 2 Group 3 Adhesion score 3.4 ±0.699 1.333 ± 1.231 0.4 ± 0.699

Example 12 Application of the Disulfide-Bond Cross-Linked HyaluronicAcid Hydrogel in the Osteoarthritis Visco-Supplement Treatment

The rabbit arthritis model reported by Mihara et al. (Osteoarthritis andCartilage 15: 543-549, 2007) was used. The process is briefly describedas follows: the rabbit was anesthetized by intramuscular injection ofketamine (35 mg/kg) and toluolzosin (5 mg/kg). The rabbit's left kneejoint in the side of kneecap was cut for a 2 cm of incision and then theexposed lateral collateral ligament was cut off; the end of the tendonwas cut open to expose the lateral meniscus followed by cutting 3.0-4.0mm off the middle of the lateral meniscus; the subcutaneous muscle layerand the skin layer were sutured, and about 0.2 ml ampicillin wasinjected by intramuscular injection in leg.

The rabbits after the partial resection of meniscus were divided intothree groups: Group 1 was a control group with physiological saline, andrespectively had an intra-articular injection with 0.2 ml physiologicalsaline on 0, 3, 6, 9 and 12 days after the surgery (a total of 5injections); Group 2 was a group treated with hyaluronic acid, andrespectively had an intra-articular injection with 0.2 ml commerciallyavailable hyaluronic acid solution (10 mg/ml) on 0, 3, 6, 9 and 12 daysafter the surgery for treatment (a total of 5 injections); Group 3 was agroup treated with the disulfide-bond cross-linked hyaluronic acidhydrogel of the present invention, and had one intra-articular injectionwith the hydrogel prepared in Example 6 (having a concentration ofhyaluronic acid of 10 mg/ml and a degree of mercapto-modification of1.54%) on 0 day after the surgery (a total of 1 injection); The painindex was measured for postoperative knee on 0, 2, 5, 8, 11 and 14 daysafter the surgery, with the pain index characterized by the weightdistribution of the left hindfoot (Mihara et al., Osteoarthritis andCartilage 15: 543-549, 2007); the rabbits were killed 15 days later, andthe appearance and histological of the postoperative knee damage wasevaluated.

The appearance and histological evaluation of the postoperative kneedamage indicated that the disulfide-bond cross-linked hyaluronic acidhydrogel of the present invention had an equivalent protective effect onthe postoperative knee to the group treated with hyaluronic acid, butwas significantly better than the control group with physiologicalsaline. The weight distribution of the left hindfoot indicated that 8,11 and 14 days after the surgery the treated group (Group 3) of thedisulfide-bond cross-linked hyaluronic acid hydrogel of the presentinvention was significantly better than the physiological saline controlgroup (Group 1) (p<0.05); while at all the postoperative observationtime points the group treated with hyaluronic acid (Group 2) had nostatistically significant difference in effects from the control groupwith physiological saline (Group 1) (p>0.05), and the group (Group 3)treated with the disulfide-bond cross-linked hyaluronic acid hydrogel ofthe present invention had no statistically significant difference ineffects from the group treated with hyaluronic acid (Group 2) (p>0.05)(see FIG. 1).

The above results indicate that the disulfide-bond cross-linkedhyaluronic acid hydrogel of the present invention has significanteffects in the osteoarthritis visco-supplement treatment, with one kneeinjection able to achieve the equivalent efficacy of five kneeinjections with the non-cross-linked hyaluronic acid.

Example 13 Preparation and Characterization of the Drug-ContainingDisulfide-Bond Cross-Linked Hyaluronic Acid Hydrogel

In the preparation process of the disulfide-bond cross-linked hyaluronicacid hydrogel of Example 6 (having a concentration of hyaluronic acid of10 mg/ml and a degree of mercapto-modification of 3.97%), 0.1-10 mgcortical hormones (e.g. Beclomethasone, Beclomethasone dipropionate,Budesonide, Dexamethasone, Prednisolone, and Prednisone) were addedrespectively to make the cortical hormones uniformly dispersed in theprepared cross-linked hydrogel.

10 ml phosphate buffer solution was added to 0.2 ml of the abovedrug-containing cross-linked hydrogel placed into a 15 ml plasticcentrifugal tube. Then centrifugal tube was placed in a shaker (37, 100rpm), and the ultraviolet absorption of the drugs in the supernatant wasmeasured at regular intervals. The measurement wavelengths were asfollows: Beclomethasone 246 nm, Beclomethasone dipropionate 240 nm,Budesonide 248 nm, Dexamethasone 242 nm, Prednisolone 248 nm, andPrednisone 244 nm.

TABLE 7 The cumulative release percentage of the drugs at different timepoints Beclo- Time Beclo- methasone Bu- Dexa- Pred- Pred- (day)methasone dipropionate desonide methasone nisolone nisone  7 68% <1% 26%41%  95%  86% 14 87% <1% 43% 63% 100%  99% 21 94% <1% 61% 75% 100% 100%

It can be seen from the results in the above Table 7 that thedisulfide-bond cross-linked hyaluronic acid hydrogel is a good drugsustained-release carrier, having good sustained release effects for thesix cortical hormones. Due to the difference in hydrophobicity of thedrugs, the release behaviors of the drugs from the hydrogel are verydifferent. The stronger the hydrophobicity of the drug is, the moresustained the release is. For example, the more hydrophilic Prednisolonewas released basically completely in 7 days; while for the veryhydrophobic Beclomethasone dipropionate, release was rarely detected.

The applications of the disulfide-bond cross-linked biocompatiblemacromolecule material of the present invention in medicine include thefollowing aspects: it is capable of promoting wound healing, it can beused as wound dressing for skin or other wounds; it can also be used forpreventing adhesion, including the fibrous adhesion between tissues ororgans after the surgery (e.g. a sinusitis surgery); it can also be usedin the osteoarthritis visco-supplement treatment as a knee lubricant.

The applications of the disulfide-bond cross-linked biocompatiblemacromolecule material prepared by the present invention in pharmacyinclude that it can be used as a sustained-release carrier for variousactive therapeutic substances to realize sustained release. The activetherapeutic substances may be chemical drugs or biologically activefactors, including antiphlogistics, antibiotics, analgesics,anaesthetics, wound healing promotors, cell growth promoters orinhibitors, immune stimulants, antiviral drugs, etc.

What is claimed is: 1-28. (canceled)
 29. A mercapto-modifiedbiocompatible macromolecule derivative with a low degree ofmercapto-modification, the mercapto-modified biocompatible macromoleculederivative comprises at least three mercapto groups in its side chain,and have a degree of mercapto-modification≦4.5%; the mercapto-modifiedbiocompatible macromolecule derivative refers to a derivative obtainedby chemically introducing the mercapto group into the side-chain groupof the biocompatible macromolecule; the degree of mercapto-modificationrefers to a percentage of the amount of the introduced mercapto group inthe amount of the available side-chain group of the biocompatiblemacromolecule for modification; and the biocompatible macromoleculerefers to a macromolecule having good biocompatibility, includingpolysaccharides, proteins, and synthetic macromolecules.
 30. Themercapto-modified biocompatible macromolecule derivative of with a lowdegree of mercapto-modification according to claim 29, wherein thedegree of mercapto-modification is 0.5%-3.0%.
 31. The mercapto-modifiedbiocompatible macromolecule derivative with a low degree ofmercapto-modification according to claim 30, wherein the degree ofmercapto-modification is 0.75%-2.5%.
 32. The mercapto-modifiedbiocompatible macromolecule derivative with a low degree ofmercapto-modification according to claim 29, wherein: thepolysaccharides are chondroitin sulfate, dermatan, heparin, heparan,alginic acid, hyaluronic acid, dermatan sulfate, pectin, carboxymethylcellulose, chitosan and carboxymethyl chitosan, or the salts andderivatives thereof; the synthetic macromolecules are polyacrylic acid,polyaspartic acid, polytartaric acid, polyglutamic acid and polyfumaricacid, or the salts and derivatives thereof; and the proteins includecollagen, alkaline glutin, acidic glutin, elastin, core protein,polysaccharide laminin and fibronectin, or the salts and derivativesthereof.
 33. The mercapto-modified biocompatible macromoleculederivative with a low degree of mercapto-modification according to claim29, wherein the biocompatible macromolecule is chondroitin sulfate,heparin, heparan, alginic acid, hyaluronic acid, polyaspartic acid,polyglutamic acid, chitosan, carboxymethyl chitosan, alkaline glutin andacidic glutin, or the salts and derivatives thereof.
 34. Themercapto-modified biocompatible macromolecule derivative with a lowdegree of mercapto-modification according to claim 33, wherein thebiocompatible macromolecule is chondroitin sulfate and hyaluronic acid,or the salts and derivatives thereof.
 35. The mercapto-modifiedbiocompatible macromolecule derivative with a low degree ofmercapto-modification according to claim 29, wherein the biocompatiblemacromolecule has a molecular weight in a range of 1,000-10,000,000. 36.The mercapto-modified biocompatible macromolecule derivative with a lowdegree of mercapto-modification according to claim 35, wherein thebiocompatible macromolecule has a molecular weight in a range of10,000-3,000,000.
 37. The mercapto-modified biocompatible macromoleculederivative with a low degree of mercapto-modification according to claim36, wherein the biocompatible macromolecule has a molecular weight in arange of 20,000-1,500,000.
 38. A disulfide-bond cross-linkedbiocompatible macromolecule material manufactured from one or more ofthe mercapto-modified biocompatible macromolecule derivatives with a lowdegree of mercapto-modification according to claim
 29. 39. Thedisulfide-bond cross-linked biocompatible macromolecule materialaccording to claim 38, wherein the material includes film and sponge ina solid form.
 40. The disulfide-bond cross-linked biocompatiblemacromolecule material according to claim 38, wherein the material ishydrogel.
 41. The disulfide-bond cross-linked biocompatiblemacromolecule material according to claim 40, wherein the hydrogel haswater content of more than 95%, which is a weight/volume percentage. 42.The disulfide-bond cross-linked biocompatible macromolecule materialaccording to claim 41, wherein the hydrogel has water content of morethan 98%, which is a weight/volume percentage.
 43. The disulfide-bondcross-linked biocompatible macromolecule material according to claim 40,wherein the hydrogel has dynamic viscosity greater than 10,000 mPa·s.44. The disulfide-bond cross-linked biocompatible macromolecule materialaccording to claim 43, wherein the hydrogel has dynamic viscositygreater than 25,000 mPa·s.
 45. The disulfide-bond cross-linkedbiocompatible macromolecule material according to claim 44, wherein thehydrogel has dynamic viscosity greater than 40,000 mPa·s.
 46. Thedisulfide-bond cross-linked biocompatible macromolecule materialaccording to claim 38, wherein the material further contains one or morepolysaccharides, proteins, synthetic macromolecules and activeingredients.
 47. The disulfide-bond cross-linked biocompatiblemacromolecule material according to claim 46, wherein thepolysaccharide, protein and synthetic macromolecule are chondroitinsulfate, heparin, heparan, alginic acid, hyaluronic acid, polyasparticacid, polyglutamic acid, chitosan, carboxymethyl chitosan, collagen,alkaline glutin and acidic glutin, or the salts and derivatives thereof.48. The disulfide-bond cross-linked biocompatible macromoleculecross-linked material according to claim 47, wherein the polysaccharide,protein and synthetic macromolecule are sodium hyaluronate, chondroitinsulfate, heparin sodium, alkaline glutin and acidic glutin.
 49. Thedisulfide-bond cross-linked biocompatible macromolecule materialaccording to claim 48, wherein the polysaccharide, protein and syntheticmacromolecule are sodium hyaluronate, chondroitin sulfate and heparinsodium.
 50. The disulfide-bond cross-linked biocompatible macromoleculematerial according to claim 46, wherein the active ingredients can beeither dispersed in the cross-linked material in a solid particle form,or dissolved in the cross-linked material.
 51. The disulfide-bondcross-linked biocompatible macromolecule material according to claim 46,wherein the active ingredients include steroids, antibiotics, antitumordrugs and various peptides protein drugs.
 52. The disulfide-bondcross-linked biocompatible macromolecule material according to claim 51,wherein the active ingredients are cortical hormones, which includebeclomethasone, beclomethasone propionate, budesonide, dexamethasone,prednisolone, and prednisone.
 53. A method of manufacturing a medicamentcomprising the disulfide-bond cross-linked biocompatible macromoleculematerial according to claim 38, the medicament selected from the groupconsisting of a postoperative adhesion prevention formulation, anosteoarthritis visco-supplement treatment formulation, and asustained-release carrier of active therapeutic substances.
 54. The useaccording to claim 53, wherein the active therapeutic substances arechemical drugs or biologically active factors.
 55. The use according toclaim 54, wherein the active therapeutic substances are antiphlogistics,antibiotics, analgesics, anaesthetics, wound healing promotors, cellgrowth promoters or inhibitors, immune stimulants, or antiviral drugs.